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Method for Production of a Bioengineered Form of Tissue Plasminogen Activator

Inactive Publication Date: 2009-10-01
AVESTHAGEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0013]Correspondingly provided by the invention are novel methods for the production of useful polypeptides comprising cultured growth of such transformed host cells particularly mammalian cells under cond

Problems solved by technology

Bolus administration could further improve the lytic rate by quickly exposing the target clot to a higher concentration of the enzyme, but single bolus administration of natural or wild type (wt) t-PA cannot be generally used, due its clearance rate.

Method used

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  • Method for Production of a Bioengineered Form of Tissue Plasminogen Activator
  • Method for Production of a Bioengineered Form of Tissue Plasminogen Activator
  • Method for Production of a Bioengineered Form of Tissue Plasminogen Activator

Examples

Experimental program
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Effect test

example 1

[0025]DNA sequences encoding tissue plasminogen activator were synthesized by de novo approach. This approach enables better codon optimization with respect to the particular mammalian cell line to be used. Further the synthetic DNA was made the subject of eucaryotic / prokaryotic expression providing isolatable quantities of polypeptides displaying biological properties of naturally occurring t-PA as well as both in vivo and invitro biological activities of t-PA.

[0026]Nucleotide sequence encoding the recombinant tissue plasminogen activator (TENECT 1) has been represented in the SEQ ID. No. 1. The codons in the coding DNA sequence of the tissue plasminogen activator that have been altered as part of the codon-optimization process to ensure optimal recombinant protein expression in mammalian cell lines such as CHO K1 and HEK 293 have been highlighted in uppercase. SEQ ID. No. 2 represents codon optimized nucleotide sequence encoding tissue plasminogen activator (TENECT 2)

[0027]Pair wi...

example 2

Verification of Authenticity of De Novo Synthesized cDNA Encoding Tissue Plasminogen Activator

[0028]The verification of the authenticity of the de novo synthesized cDNA molecules as supplied by the commercial service provider was done by automated DNA sequencing and the results obtained are depicted in FIGS. 2 & 3.

example 3

Sub-Cloning of TENECT & TENECT-Opt cDNAs into the pcDNA3.1D / V5-His Mammalian Cell-Specific Expression Vector

[0029]Subsequent to the verification of the authenticity of the de novo synthesized cDNA molecules (TENECT & TENECT-Opt) by automated DNA sequencing as shown above. TENECT & TENECT-Opt were individually sub-cloned into the mammalian cell-specific expression vector pcDNA3.1D / V5-His to generate the transfection-ready constructs. The details of the procedures used are given below:

[0030]A. Reagents and enzymes:[0031]1. QIAGEN gel extraction kit & PCR purification kit[0032]2. pcDNA 3.1D / V5-His vector DNA (Invitrogen)

EnzymeSupplierU / μl10x buffer1. BamHIBangalore Genei10Buffer E2. XhoIBangalore Genei10Buffer E3. HindIIIBangalore Genei20Buffer E4. XhoIBangalore Genei10Buffer E5. T4 DNA ligaseBangalore Genei40Ligase Buffer

All reactions were carried out as recommended by the manufacturer. For each reaction the supplied 10× reaction buffer was diluted to a final concentration of 1×.

[0033...

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Abstract

The present invention relates to the recombinant method used for the production of soluble form of human tissue plasminogen activator variant. In this variant the threonine at position 103 of the endogenous tissue plasminogen activator is replaced by an asparagine leading to a new glycosylation site. At position 117 of the endogenous tissue plasminogen activator asparagine has been replaced by glutamine, leading to the removal of an N linked glycosylation site. At position 296-299 the amino acids lysine, histidine, arginine, and arginine have been replaced by four alanine amino acids. The invention further relates to the de novo synthesis of the nucleic acid sequence encoding tissue plasminogen activator, transformation of the constructed nucleic acid sequences into competent bacteria and sub-cloning of the same into mammalian expression vectors for the expression of the desired protein. DNA constructs comprising the control elements associated with the gene of interest have been disclosed. The recombinant human tissue plasminogen activator, according to the invention, and the salts and functional derivatives thereof, may comprise the active ingredient of pharmaceutical compositions for treatment of treatment of heart attack and stroke patients. These compositions are yet another aspect of the present invention.

Description

FIELD OF INVENTION[0001]The present invention relates to the recombinant method used for the production of soluble form of human tissue plasminogen activator variant. In this variant the threonine at position 103 of the endogenous tissue plasminogen activator is replaced by an asparagine leading to a new glycosylation site. At position 117 of the endogenous tissue plasminogen activator asparagine has been replaced by glutamine, leading to the removal of an N linked glycosylation site. At position 296-299 the amino acids lysine, histidine, arginine, and arginine have been replaced by four alanine amino acids.[0002]The invention further relates to the de novo synthesis of the nucleic acid sequence encoding tissue plasminogen activator, transformation of the constructed nucleic acid sequences into competent bacteria and sub-cloning of the same into mammalian expression vectors for the expression of the desired protein.[0003]DNA constructs comprising the control elements associated with...

Claims

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Application Information

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IPC IPC(8): A61K38/48C12N15/09A61P35/00
CPCC12Y304/21069C12N9/6459A61P35/00A61P7/02C12N9/6456C12N15/67A61K38/00
Inventor PATELL, VILLOO MORAWALA
Owner AVESTHAGEN
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